This project proposes to gain an understanding of the physiological roles of and mechanisms by which certain eukaryotic "unwinding" proteins destabilize nucleic acid helices, in particular RNA secondary structure. Several of these proteins accelerate conformational changes of transfer RNA and 55 RNA molecules which normally exhibit large activation barriers. Nucleic acid helix destabilizing proteins are being isolated and characterized from single-cell organisms such as yeast. We eventually hope to obtain mutants to unwinding proteins, which will be a significant step toward establishing the in vivo function(s) of these factors. Another approach we are pursuing to this end is the isolation and characterization of nucleic acid binding proteins from subcellular mammalian fractions, with emphasis on the nucleocytoplasmic interface. The isolated proteins' interaction with nucleic acids is being characterized by both physical and chemical techniques. Using ultraviolet spectrophotometry, we routinely determine the melting temperature, Tm, of helical nucleic acids in the presence of these proteins. Circular dichroism has been very useful in determining binding constants. These and other methods are being utilized to establish the range of binding strengths and specificities of isolated proteins, as well as the molecular mechanisms of protein-induced nucleic acid "unwinding."